CN100415769C - Oligomeric or polymerized methylene protein and its separation and purification process and use - Google Patents
Oligomeric or polymerized methylene protein and its separation and purification process and use Download PDFInfo
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Abstract
The present invention relates to an oligomer or a poly-ylidene protein, a method and the application in the process of separating and purifying oligomer or poly-ylidene protein. The method can protect the activity of oligomer or poly-ylidene protein, and can improve yield. The oligomer protein or the poly-ylidene protein provides protein comprising more than two independent ylidenes polymerized by intermolecular force. The method comprises: a. protective agent of which the concentration is from 0.5 to 40% weight/volume is added in cell culture fluid, animal or plant tissue extract solution, or supernatant liquid after cell breakage, balanced solution and eluent solution at normal temperature; b. the method carries out cell breakage, membrane separation, adsorption or chromatographic separation and purification by conventional technology in order to obtain the oligomer protein or the poly-ylidene protein. The protein is applied in the process for preparing genetic engineering protein or vaccine. The method can effectively protect the integrity of protein molecular structures, reduce biological activity loss caused by separation and purification and improve target protein yield.
Description
Technical field
The present invention relates to proteinic preparing technical field, particularly a kind of oligomerization or polymerized methylene protein and in the process of separation and purification oligomerization or polymerized methylene protein can protect its activity can improve the method and the purposes of yield again.
Background technology
In the proteinic production of genetically engineered drug, low the hanging down with purity of yield is the key issue that needs to be resolved hurrily.Here except the biological composition complexity, be difficult to separate, the active forfeiture of many target proteins self also is a major reason.Because having to improve slightly, the market value of pharmaceutical protein costliness, the rate of recovery just might bring huge economic benefit, so inactivation and the countermeasure thereof of protein involved in separation and purification received great concern in recent years.
Oligomerization or polymerized methylene protein refer to by the polymer of subunit independent of each other more than two by interacting and combine between the molecule.Wherein, each subunit generally is made up of a peptide chain, but also has two above peptide chain warp disulfide linkage to be connected to form.Each subunit self is folded into certain sterie configuration, relies on hydrophobic interaction, hydrogen bond, ionic linkage and reactive force to flock together between the different then subunits, forms proteinic quaternary structure, interacts between the subunit to constitute the integral body of chemical-biological activities.At occurring in nature, this protein of being made up of oligomerization (2,3,4 etc.) and poly (tens to up to a hundred) subunit is quite a lot in whole protein families.For example, oxyphorase is made up of 2 α and 2 β subunits, and tetramer structure has determined its combination and the characteristic that discharges oxygen.Human tumor necrosis factor is made of three subunits, and the molecular weight of each subunit is 1.7 ten thousand, and the molecular weight of whole molecule is 5.1 ten thousand.Luci also is a tripolymer, and its activity is by the tripolymer structures shape.Many viruses contain the polymer structure, are the spirochete of a polymerized methylene as tobacco in tikka virus, 16.3 subunits of every circle, and pitch is 2.3nm.Vaccine by the virion transformation also usually is the subunit polymer, is the spheroid that 2.0 ~ 3.0 ten thousand albumen is formed as hepatitis B vaccine (HBsAg) by a plurality of molecular weight, and molecular weight is about about 2,000,000.
The biologic activity of oligomerization or polymerized methylene protein is usually relevant with the structure of its aggregate.The tetramer structure of oxyphorase has determined that its oxygenate curve is the S type; Independent subunit or α β dimer then do not possess this specific character.The trimerical biologic activity of human tumor necrosis factor TNF-alpha is the highest, when being each subunit Individual existence active 8 times.Alcohol Dehydrogenase from Yeast has four subunits, and each molecular weight is 4.5 ten thousand, and single subunit has only four subunit combinations just to have activity without any catalytic activity together.
The difficult point of separation and purification oligomerization or polymerized methylene protein is how to prevent dissociating of subunit.Reactive force between the subunit comprises hydrophobic interaction, hydrogen bond, ionic linkage, Van der Waals force, belongs to weak interaction.Generally about 5-15kcal/mol, conformational stability is very low for their stabilized free energy Δ Gstab, and this has report [Pace, C.N., Trends Biochem.Sci.1990,15:14-17] in " biological chemistry trend " magazine.In the separation and purification process, stirring, temperature rise, pH change, salt concn changes, add tensio-active agent or organic solvent etc. is result and the necessary operation that separation and purification is difficult to avoid, but these results and operation usually cause dissociating and structural changes of subunit.Can cause the decline of the rate of recovery on the one hand, also can cause the biologic activity forfeiture on the other hand.The protein of inactivation just becomes impurity, and is not only useless, also may be poisonous.
About the stability in the protein soln a lot of researchs have been arranged, from molecular structure, the general character of the various low unit alcohol that water soluble hydroxy compound such as this area researchist are familiar with, poly-low unit alcohol, monose, disaccharides, oligosaccharides and polysaccharide is hydroxyl and the ether that contains different quantities, have good hydrophilicity, can coexist as in the solution with protein.But when they existed, the thermodynamic property of solution then changed.To this, Timasheff is with preferentially getting rid of theoretical (Timasheff, S.N., in Stability of Protein Pharmaceuticals Part B, Ahern, T.J.and Manning, M.C. (eds.), New York, Plenum Press, 1992,265-285) explain, think that hydrophilic polymer molecules and protein molecule resemble the polyoxyethylene glycol all preferentially combines with molecule and avoid being bonded to each other in solution.Because fight for water molecules each other, and water molecules forms ordered arrangement around them, the free energy height of solution will be so protein molecule will reduce its surface as far as possible.Keep stable compact form.Under compact form, protein is the most stable, is difficult for recurring structure and changes.Therefore, resemble glycerol, the preservatives when polyoxyethylene glycol, the glucose in the monose in poly-low first alcohol all are used as protein lyophilize and storage that hangs down in first alcohol.For example, Chinese patent application 96195830, a kind of stable lyophilized protein formulation of U.S. Geneart AG (Genentech) application protection wherein just contains the water soluble hydroxy compound.Chinese patent application 00109612, the preparation production method of Shenyang three lives pharmacy stock company application protection recombined human thrombopoietin preparation, the final finished product protection agent that adds also contains the water soluble hydroxy compound.The United States Patent (USP), the European patent that similarly are used for the lyophilize of protein formulation and preservation are also a lot.
But up to now, from stock liquid (cell culture fluid, tissue extract) during separation and purification protein how the structure of stabilizing protein do not change, the research of this respect is also seldom; To hang down in the protein separation of not seeing as yet unit alcohol, poly-low unit alcohol, monose, disaccharides, oligosaccharides and polysaccharide as protective material, make it to follow partly or the report of whole separation and purification process; Particularly, how to avoid subunit dissociating in the separation and purification process also not see any report to oligomerization or polymerized methylene protein.
Summary of the invention
The objective of the invention is to: in separation and purification oligomerization and polymerized methylene protein process, avoid proteinic structure to change; And in order in separation and purification oligomerization and polymerized methylene protein process, to improve the product rate of recovery; Thereby a kind of oligomerization or polymerized methylene protein are provided and in the process of separation and purification oligomerization or polymerized methylene protein, can protect its activity can improve the method and the purposes of yield again.
The objective of the invention is to realize by following technical scheme:
Oligomerization provided by the invention or polymerized methylene protein are the protein that is condensed together by intermolecular forces by independent subunit more than 2; As alcoholdehydrogenase (tetramer), human tumor necrosis factor (tripolymer), human hemoglobin (tetramer), recombinant hepatitis b vaccine (many subunits aggressiveness) etc.
Separation and purification oligomerization provided by the invention or polymerized methylene protein method may further comprise the steps:
(1) at normal temperatures, adding weight/volume in cytoclasis supernatant liquor, balance liquid and elutriant is the water soluble hydroxy compound protective material of 0.5-40%;
(2) technology is carried out membrane sepn and chromatographic separation purifying routinely, thereby obtains oligomerization or polymerized methylene protein.
Wherein protective material is the water soluble hydroxy compound, and this water soluble hydroxy compound comprises: monohydroxy-alcohol such as methyl alcohol, ethanol, propyl alcohol; Dibasic alcohol such as ethylene glycol, propylene glycol, butyleneglycol; Trivalent alcohol such as glycerol; The multipolymer of diol polymer such as polyoxyethylene glycol, polypropylene glycol, ethylene glycol and propylene glycol; Poly-hydroxy monose such as glucose, fructose, seminose, wood sugar, disaccharides such as sucrose, lactose, maltose; Polysaccharide such as starch, dextran.
Oligomerization provided by the invention and polymerized methylene protein are applied to engineered protein or vaccine production process.
Protein is meant oligomerization or polymerized methylene protein metabolic or that produce with gene engineering method from animal, plant and microorganism cells.
Be used for protein separation if will hang down unit alcohol, poly-low unit alcohol, monose, disaccharides, oligosaccharides and polysaccharide, then to solve they whether with the problem of existing isolation technique compatibility, i.e. whether their adding can reduce the efficient of separation and purification.This problem is resolved in the present invention.According to the present invention, smudge cells (Cell disruption, Cell disintegration), membrane sepn (Membrane separation), absorption (Adsorption) and chromatography (Chromatography) can with above-mentioned protective material compatibility.
Low unit alcohol, poly-low unit alcohol, monose, disaccharides, oligosaccharides and polysaccharide can be used alone, but also also mix together.To some protein, mix the better effects if of using.
For different target proteins,, can both reach the dual purpose of protected protein matter activity and separation and purification effect by adding and regulating protectant kind, molecular weight and concentration.Though the effective protectant kind for different proteins is usually different, in general, the glycerol effect in low first alcohol is better; Following effect structure is better in poly-low first alcohol:
HO-(CH
2-CH
2-O)
n-(CH
2-CH
2-O)
m-H
N and m are the integer of 0-200, and n+m is more than or equal to 1.In fact this comprises polyoxyethylene glycol, polypropylene glycol or both mixtures.In the monose in glucose, the disaccharides in sucrose, the polysaccharide effect of poly-dextrose better.
The concentration of protective material in protein soln is extremely important.Different proteins required protective material concentration difference in different purification procedures, its optimum range usually is a 0.5-40% weight/volume (W/V).It is poor that concentration is crossed low protection effect, and excessive concentration causes proteinic precipitation easily.
Method of the present invention has following advantage and effect:
This method comprises that with the water soluble hydroxy compound be protective material; in carrying out from cell culture fluid, animal or plant tissue extract the tight basic proteinic process of separation and purification oligomerization or poly; by protectant kind and concentration in interpolation and control nutrient solution or the extracting solution; cracking does not take place in the oligomerization of protected protein matter or polymerized methylene; thereby protect this proteinic biologic activity, also satisfy these proteinic needs of separation and purification simultaneously.This method is applied to engineered protein or vaccine production process as: extraction cell interior material, with vary in size molecule and with angular spectrum chromatography fractionation by adsorption difference biomacromolecules etc. of membrane sepn; the effective integrity of the molecular structure of protected protein matter; reduce the separation and purification operation to the loss that its biologic activity causes, the yield of target protein is improved.For different target proteins,, can reach the dual purpose of protected protein matter activity and separation and purification effect by regulating protectant kind, molecular weight and concentration.
Portion water dissolubility oxy-compound involved in the present invention has been applied to the lyophilize and the preservation of protein formulation by forefathers, but is not to be added in the initial biological extraction liquid protective material as the separation and purification process as protective material.As previously mentioned, resemble glycerol, the preservatives when polyoxyethylene glycol, the glucose in the monose in poly-low first alcohol all are used as protein lyophilize and storage that hangs down in first alcohol.
But will hang down in the protein separation of not seeing as yet up to now, unit alcohol, poly-low unit alcohol, monose, disaccharides, oligosaccharides and polysaccharide as protective material, make it to follow partly or patent, paper or the report of whole separation and purification process; That is to say; the present invention is carrying out from cell culture fluid; the process of separation and purification oligomerization or polymerized methylene protein in the animal or plant tissue extract (comprises extraction cell interior material; with membrane sepn vary in size molecule and with processes such as the different biomacromolecules of chromatography fractionation by adsorption) in; by protectant kind and concentration in interpolation and control nutrient solution or the extracting solution; cracking does not take place in the oligomerization of protected protein matter or polymerized methylene; thereby protect this proteinic biologic activity; also satisfy simultaneously these proteinic needs of separation and purification; reduce the separation and purification operation to the loss that its biologic activity causes, the yield of target protein is improved.
Description of drawings:
Fig. 1: add PEG (1%) or do not add the comparative studies figure of PEG to anionic exchange medium DEAE-Sepharose CL 6B adsorption and purification rhTNF-α process influence.
The PEG concentration: 1%, adsorption column: 16 * 55mm, inlet amount: 10mg/ml medium.The gross activity of rhTNF-α in the feed liquid: 5.0 * 10
7u
Embodiment
Embodiment 1 extracts ethanol dehydrogenase (tetramer protein) from yeast
Yeast: commercially available dry bread yeast (Dutch Gist-Brocades yeast company limited) is dissolved in the distilled water, and yeast concn is 10% weight/volume (W/V) (dry weight), is mixed with 8 liters of yeast suspensions.Stir one hour down with the activation yeast at 4 ℃.
Add protective material: after one hour, cell suspending liquid is divided into two parts (each 4 liters), under agitation, the yeast suspension after a copy of it activation adds the mixing solutions of 1 liter of glycerine and 20% (W/V) sucrose, and making suspension vol is 5 liters; Another part cell suspending liquid does not add any protective material, and replenishing with distilled water is that 5 liters of conducts are with reference to comparing.
Smudge cells: the equal stock pump of high pressure that adopts U.S. APV Manton Gaulin company to produce, join five liters of stock liquids in the head tank and stir, note regulating stirring intensity, do not make solution sneak into a large amount of bubbles, influence the efficient of high pressure homogenate broken cell because stir too violent.The mixing speed that this experiment is adopted is 150rpm.After starting the equal stock pump of high pressure, it is 60Mpa that average rate is regulated pressure, connects a collector and collect broken cell slurries pump discharge outside, and collector places under 4 ℃ of coolings, wait to collect whole slurries after, again slurries are led repeat back into charging basket broken with the raising cell crashing ratio.Broken repeatedly number of times is 4 times.
Activation analysis: broken back cell slurries are in 4 ℃, and 15, centrifugal 15min removes cell debris under the 000g, gets supernatant liquor and does enzyme activity assay.Coomassie brilliant blue method [Bradford is adopted in the protein concn analysis, M.M.1976.Anal.Biochem.72:248-254], with Coomassie brilliant blue G250 is reagent, under acidic conditions, form mixture with protein, under 595nm, maximum absorption band is arranged, absorbancy and protein concn ratio make typical curve with bovine serum albumin as standard protein.The ethanol dehydrogenase analysis adopts nadide to generate behind the nadide of going back ortho states the feature that is absorbed as at the 340nm place after oxidation of ethanol is acetaldehyde, as alcoholdehydrogenase activity measurement (work such as Jiang Chuankui, " vitality test of toolenzyme ", Shanghai science tech publishing house, 1982).
Experimental result: the vigor that does not add the resulting ethanol dehydrogenase of any protective material is 25u/ml, and wherein u represents enzyme activity unit, and/ml represents the slurries after every ml cells fragmentation.Under same experiment condition, after adding glycerine and the sucrose, the vigor of resulting ethanol dehydrogenase is 40u/ml, than not improving 60% with protective material.
Embodiment 2 extracts human tumor necrosis factor (tripolymer) from gene engineering colibacillus
Intestinal bacteria (E.coli cell) are cultivated: recombinant human tumor necrosis factor (rhTNF-α) intestinal bacteria JM103, substratum is 1.5% weight/volume (W/V) Tryptones, 1.3% weight/volume (W/V) yeast soaks powder, 0.25 weight/volume (W/V) NaCl, 0.2% weight/volume (W/V) glucose, the final concentration of pH7.5 tsiklomitsin are 12.5 μ g/l.The fermentation of reorganization bacterium is carried out under 37 ℃ with 6 liters of fermentor tanks (U.S. New Brunswick Scientific, Co. product, model BIOFLO II), and fermentation period is 12 hours.After the fermentation, in 4,500g, following centrifugal 20min collects the E.coli cell.Cell is through 10mmol Tris CHl, 1mmol EDTA, and the buffered soln washing of pH8.0, becoming concentration with same buffered soln suspension cell again is the cell suspending liquid of 20wt%.
Add protective material: the cell suspending liquid of 20wt% is divided into 4 parts, adds polyoxyethylene glycol (PEG) 200,1000,4000 respectively, last portion does not add any protective material as reference.Polyoxyethylene glycol concentration in first three part suspension is: 15% weight/volume (W/V); 10wt% (W/V), 4wt% (W/V).
Smudge cells: high speed water flat ball mill (Dyno-Mill, Switzerland WAB company product are adopted in cytoclasis.) the bead loading amount is 90% cavity volume.Operational condition is 3 ℃, 2500r.p.m, 3min.
Activation analysis: broken back cell slurries are in 4 ℃, and 15, centrifugal 15min removes cell debris under the 000g, gets supernatant liquor and does biological this activation analysis.The TNF activation analysis is with reference to people's such as Aggarwal method [Aggarwal, B.B., Kohr, w.j., Hass, P.E., Moffar, B., Spence, S.A., Henzel, W.J., Bringman, T.S., Nedwin, G.E., Goeddel, D.V.and Harkins, R.N.J.Biol.Chem.260,2345-2354,1985.] carry out.The protein concn analysis is carried out [Bradford, M.M.1976.Anal.Biochem.72:248-254] according to the Bradford method, with bovine serum albumin as standard protein.
Experimental result: the rhTNF-alpha active of the slurries behind four parts of smudge cellses is as follows:
As can be seen from the above table, add protective material the RhTNF-alpha active is improved more than 30%, wherein the effect of PEG200 is best, makes active raising more than 2 times.
Embodiment 3 purification via adsorption-based process genetically engineered human tumor necrosis factors
Absorption: with anionic exchange medium DEAE-Sepharoes C1-6B pack into adsorption column (16 * 55mm), use 0.01MTris-HCl, the abundant balance of pH8.0 (containing or do not contain polyoxyethylene glycol PEG).With concentration is in 1wt% polyoxyethylene glycol PEG200, the 600 or 4000 cytoclasis supernatant liquor that joins no polyoxyethylene glycol PEG respectively and exist, balance liquid, the elutriant.Charging is removed the protein that is not adsorbed to baseline with level pad drip washing, uses 75mM NaCl, and 10mM Tris-HCl carries out stepwise elution, collects each elution peak and measures protein concn and the activity of RhTNF-α.
Activation analysis: the TNF activation analysis is with reference to people's such as Aggarwal method [Aggarwal, B.B., Kohr, w.j., Hass, P.E., Moffar, B., Spence, S.A., Henzel, W.J., Bringman, T.S., Nedwin, G.E., Goeddel, D.V.and Harkins, R.N.J.Biol.Chem.260,2345-2354,1985.] carry out.The protein concn analysis is carried out [Bradford, M.M.1976.Anal.Biochem.72:248-254] according to the Bradford method, with bovine serum albumin as standard protein.
Experimental result: Fig. 1 has shown adding PEG200, PEG600, PEG4000 or has not added PEG to DEAE-SepharoseCL 6B adsorption and purification rhTNF-α process influence.The result shows, do not having PEG to have that activity recovery only is 65%, the purifying multiple is 7.When adding the PEG of different molecular weight in the feeding liquid, the rate of recovery and purifying multiple all increase.PEG200 and PEG4000 then make the rate of recovery surpass or reach 100%, illustrate that a small amount of product that changes has recovered activity.
Embodiment 4 hydrophobic interaction chromatographies prepare dephosphorization fat bovine hemoglobin
Hydrophobic interaction chromatography: hydrophobic interaction chromatography medium agarose-6B type packed into, and (130mm * 16mm) is with the abundant balance of 0.01M phosphate buffered saline buffer (containing or do not contain PEG) for chromatography column.PEG4000 or PEG10000 are joined in the erythroclasis liquid, balance liquid, elutriant of no PEG.Charging goes out oxyphorase with elutriant drip washing, collects the back and measures protein concn and phospholipids content.
Protein concn is analyzed: according to the Bradford method carry out to the greatest extent (Bradford, M.M.1976.Anal.Biochem.72:248-254)
Experimental result:
Table 2-3 compares with the effect of the PEG phosphoric acid buffer wash-out of Different Weight volumetric concentration and molecular weight
As can be seen from the above table, hydrophobic interaction chromatography has improved more than 3 times the rate of recovery of oxyphorase at the adding protective material, and the removal of phosphatide is not influenced.
Claims (3)
1. the method for oligomerization or polymerized methylene protein separation and purification is characterized in that: may further comprise the steps:
(1) at normal temperatures, adding weight/volume in cytoclasis supernatant liquor, balance liquid and elutriant is the water soluble hydroxy compound protective material of 0.5-40%;
(2) technology is carried out membrane sepn and chromatographic separation purifying routinely, thereby obtains oligomerization or polymerized methylene protein.
2. the method for oligomerization as claimed in claim 1 or polymerized methylene protein separation and purification is characterized in that: described water soluble hydroxy compound protective material is: monohydroxy-alcohol, dibasic alcohol, trivalent alcohol, diol polymer, poly-hydroxy monose, disaccharides, oligosaccharides or polysaccharide use separately or mix and use.
3. the method for separation and purification oligomerization as claimed in claim 2 or polymerized methylene protein is characterized in that: described monohydroxy-alcohol is methyl alcohol or ethanol; Dibasic alcohol is ethylene glycol, propylene glycol or butyleneglycol; Trivalent alcohol is a glycerol; Diol polymer is the multipolymer of polyoxyethylene glycol, polypropylene glycol or ethylene glycol and propylene glycol; Poly-hydroxy monose is glucose, fructose, seminose or wood sugar; Disaccharides is sucrose, lactose or maltose; Polysaccharide is starch or dextran.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN85103552A (en) * | 1985-05-04 | 1986-10-29 | 细胞技术有限公司 | Process by the nutrient solution separating animal cells |
| CN1186119A (en) * | 1996-12-25 | 1998-07-01 | 吴海燕 | Recombinative human thrombocytopoietic factor and production thereof |
| CN1191490A (en) * | 1995-07-27 | 1998-08-26 | 基因技术股份有限公司 | Stable isotonic lyophilized protein formulation |
| CN1196956A (en) * | 1997-04-09 | 1998-10-28 | 杜菲尔国际开发有限公司 | Influenza vaccine |
| WO2000049892A2 (en) * | 1999-02-22 | 2000-08-31 | Centro De Histoterapia Placentaria | Dietetic supplement from human placenta |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN85103552A (en) * | 1985-05-04 | 1986-10-29 | 细胞技术有限公司 | Process by the nutrient solution separating animal cells |
| CN1191490A (en) * | 1995-07-27 | 1998-08-26 | 基因技术股份有限公司 | Stable isotonic lyophilized protein formulation |
| CN1186119A (en) * | 1996-12-25 | 1998-07-01 | 吴海燕 | Recombinative human thrombocytopoietic factor and production thereof |
| CN1196956A (en) * | 1997-04-09 | 1998-10-28 | 杜菲尔国际开发有限公司 | Influenza vaccine |
| WO2000049892A2 (en) * | 1999-02-22 | 2000-08-31 | Centro De Histoterapia Placentaria | Dietetic supplement from human placenta |
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Application publication date: 20030820 Assignee: Hualan Biological Vaccine Co., Ltd. Assignor: Institute of Process Engineering, Chinese Academy of Sciences Contract record no.: 2012410000079 Denomination of invention: Oligomeric or polymerized methylene protein and its separation and purification process and use Granted publication date: 20080903 License type: Exclusive License Record date: 20121016 |
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